Genes encoding key catalyzing mechanisms for ethanol production from syngas fermentation

ABSTRACT

Gene sequences of key acetogenic clostridial species were sequenced and isolated. Genes of interest were identified, and functionality was established. Key genes of interest for metabolic catalyzing activity in clostridial species include a three-gene operon coding for CODH activity, a two-gene operon coding for PTA-ACK, and a novel acetyl coenzyme A reductase. The promoter regions of the two operons and the acetyl coA reductase are manipulated to increase ethanol production.

RELATED U.S. APPLICATION DATA

This application claims the benefit of and priority to U.S. patentapplication Ser. No. 12/336,278 filed Dec. 16, 2008 as acontinuation-in-part application. The entirety of that application isincorporated by reference herein. The content of the sequence listinginformation recorded in computer readable form is identical to thecompact disc sequence listing and, where applicable, includes no newmatter, as required by 37 CFR 1.821 (e), 1.821(f), 1.821(g), 1.825(b),or 1.825(d).

FIELD OF THE INVENTION

This invention relates to the cloning and expression of novel geneticsequences of microorganisms used in the biological conversion of CO, H2,and mixtures comprising CO and/or H2 to biofuel products.

BACKGROUND

Synthetic gas (syngas) is a mixture of carbon monoxide (CO) gas, carbondioxide (CO₂) gas, and hydrogen (H₂) gas, and other volatile gases suchas CH₄, N₂, NH₃, H₂S and other trace gases. Syngas is produced bygasification of various organic materials including biomass, organicwaste, coal, petroleum, plastics, or other carbon containing materials,or reformed natural gas.

Acetogenic Clostridia microorganisms grown in an atmosphere containingsyngas are capable of absorbing the syngas components CO, CO₂, and H₂and producing aliphatic C₂-C₆ alcohols and aliphatic C₂-C₆ organicacids. These syngas components activate Wood-Ljungdahl metabolic pathway100, shown in FIG. 1, which leads to the formation of acetyl coenzyme A102, a key intermediate in the pathway. The enzymes activatingWood-Ljungdahl pathway 100 are carbon monoxide dehydrogenase (CODH) 104and hydrogenase (H₂ase) 106. These enzymes capture the electrons fromthe CO and H₂ in the syngas and transfer them to ferredoxin 108, aniron-sulfur (FeS) electron carrier protein. Ferredoxin 108 is the mainelectron carrier in Wood-Ljungdahl pathway 100 in acetogenic Clostridia,primarily because the redox potential during syngas fermentation is verylow (usually between −400 and −500 mV). Upon electron transfer,ferredoxin 108 changes its electronic state from Fe³⁺ to Fe²⁺.Ferredoxin-bound electrons are then transferred to cofactors NAD⁺ 110and NADP⁺ 112 through the activity of ferredoxin oxidoreductases 114(FORs). The reduced nucleotide cofactors (NAD⁺ and NADP⁺) are used forthe generation of intermediate compounds in Wood-Ljungdahl pathway 100leading to acetyl-CoA 102 formation.

Acetyl-CoA 102 formation through Wood-Ljungdahl pathway 100 is shown ingreater detail in FIG. 2. Either CO₂ 202 or CO 208 provide substratesfor the pathway. The carbon from CO₂ 202 is reduced to a methyl groupthrough successive reductions first to formate, by formate dehydrogenase(FDH) enzyme 204, and then is further reduced to methyl tetrahydrofolateintermediate 206. The carbon from CO 208 is reduced to carbonyl group210 by carbon monoxide dehydrogenase (CODH) 104 through a second branchof the pathway. The two carbon moieties are then condensed to acetyl CoA102 through the action of acetyl-CoA synthase (ACS) 212, which is partof a carbon monoxide dehydrogenase (CODH/ACS) complex. Acetyl-CoA 102 isthe central metabolite in the production of C₂-C₆ alcohols and acids inacetogenic Clostridia.

Ethanol production from Acetyl CoA 102 is achieved via one of twopossible paths. Aldehyde dehydrogenase facilitates the production ofacetaldehyde, which is then reduced to ethanol by the action of primaryalcohol dehydrogenases. In the alternative, in homoacetogenicmicroorganisms, an NADPH-dependent acetyl CoA reductase (“AR”)facilitates the production of ethanol directly from acetyl CoA.

Wood-Ljungdahl pathway 100 is neutral with respect to ATP productionwhen acetate 214 is produced (FIG. 2). When ethanol 216 is produced, oneATP is consumed in a step involving the reduction of methylenetetrahydrafolate to methyl tetrahydrofolate 206 by a reductase, and theprocess is therefore net negative by one ATP. The pathway is balancedwhen acetyl-PO₄ 218 is converted to acetate 214.

Acetogenic Clostridia organisms generate cellular energy by iongradient-driven phosphorylation. When grown in a CO atmosphere, atransmembrane electrical potential is generated and used to synthesizeATP from ADP. Enzymes mediating the process include hydrogenase, NADHdehydrogenases, carbon monoxide dehydrogenase, and methylenetetrahydrofolate reductase. Membrane carriers that have been shown to belikely involved in the ATP generation steps include quinone,menaquinone, and cytochromes.

The acetogenic Clostridia produce a mixture of C₂-C₆ alcohols and acids,such as ethanol, n-butanol, hexanol, acetic acid, and butyric acid, thatare of commercial interest through Wood-Ljungdahl pathway 100. Forexample, acetate and ethanol are produced by C. ragsdalei in variableproportions depending in part on fermentation conditions. However, thecost of producing the desired product, an alcohol such as ethanol, forexample, can be lowered significantly if the production is maximized byreducing or eliminating production of the corresponding acid, in thisexample acetate. It is therefore desirable to metabolically engineeracetogenic Clostridia for improved production of selected C₂-C₆ alcoholsor acids through Wood-Ljungdahl pathway 100 by modulating enzymaticactivities of key enzymes in the pathway.

SUMMARY OF THE INVENTION

One aspect of the present invention provides novel sequences for threekey operons which code for enzymes that catalyze the syngas to ethanolmetabolic process: one coding for a carbon monoxide dehydrogenase, amembrane-associated electron transfer protein, a ferredoxinoxidoreductase, and a promoter; a second operon coding for an acetatekinase, phosphotransacetylase, and a promoter, and a third operon codingfor an acetyl CoA reductase and a promoter.

Another aspect of the invention provides an isolated vector ortransformant containing the polynucleotide sequence coding for theoperons described above.

Another aspect of the invention provides a method of producing ethanolcomprising: isolating and purifying anaerobic, ethanologenicmicroorganisms carrying the polynucleotides coding for an operoncomprising carbon monoxide dehydrogenase, a membrane-associated electrontransfer protein, a ferredoxin oxidoreductase, and a promoter; an operoncoding for an acetate kinase, phosphotransacetylase, and a promoter, oran operon coding for an acetyl CoA reductase and a promoter; fermentingsyngas with said microorganisms in a fermentation bioreactor; providingsufficient growth conditions for cellular production of NADPH, includingbut not limited to sufficient zinc, to facilitate ethanol productionfrom acetyl CoA.

Another aspect of the invention provides a method of producing ethanolby isolating and purifying anaerobic, ethanologenic microorganismscarrying the polynucleotide coding for acetyl coenzyme A reductase;fermenting syngas with said microorganisms in a fermentation bioreactor;and providing sufficient growth conditions for cellular production ofNADPH, including but not limited to sufficient zinc, to facilitateethanol production from acetyl CoA.

Yet another aspect of the present invention provides a method ofincreasing ethanologenesis or the ethanol to acetate production ratio ina microorganism containing the nucleotide sequence(s) coding for one ofmore of the operons described above, said method comprising: modifying,duplicating, or downregulating a promoter region of said nucleotidesequence to increase the activity of the Acetyl Coenzyme A reductase,said sequence being at least 98% identical to SEQ ID NO. 3, or to causeoverexpression or underexpression of the nucleotide sequence.

The present invention is illustrated by the accompanying figuresportraying various embodiments and the detailed description given below.The figures should not be taken to limit the invention to the specificembodiments, but are for explanation and understanding. The detaileddescription and figures are merely illustrative of the invention ratherthan limiting, the scope of the invention being defined by the appendedclaims and equivalents thereof. The drawings are not to scale. Theforegoing aspects and other attendant advantages of the presentinvention will become more readily appreciated by the detaileddescription taken in conjunction with the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram illustrating the electron flow pathway during syngasfermentation in acetogenic Clostridia including some of the key enzymesinvolved in the process;

FIG. 2 is a diagram illustrating the Wood-Ljungdahl (C₁) pathway foracetyl CoA production and the enzymatic conversion of acetyl-CoA toacetate and ethanol;

FIG. 3 is a diagram illustrating a genetic map containing the locationof one of the carbon monoxide dehydrogenase (CODH) operons whichincludes cooS, cooF and a ferredoxin oxidoreductase (FOR), in accordancewith the invention;

FIG. 4 is a diagram showing the amino acid alignment of the gene forNADPH dependent secondary alcohol dehydrogenase in C. ragsdalei [SEQ IDNo. 4], C. ljungdahlii [SEQ ID No. 5] and Thermoanaerobactor ethanolicus[SEQ ID No. 6], in accordance with the invention;

FIG. 5 is a diagram illustrating the Wood-Ljungdahl pathway for ethanolsynthesis and showing a strategy for specifically attenuating oreliminating acetate production in acetogenic Clostridia by knocking outthe genes encoding acetate kinase (ack) and phosphotransacetylase (pta)or by modulating acetate production by mutating or replacing thepromoter driving phosphotransacetylase and acetate kinase geneexpression, in accordance with the invention;

FIG. 6 is a diagram of the Wood-Ljungdahl pathway for ethanol synthesis,and shows a strategy for specifically increasing ethanol production inC. ragsdalei by overexpression of an acetyl CoA reductase in a hostknocked out for acetate kinase or phosphotransacetylase activity, inaccordance with the invention;

FIG. 7 is a diagram of the Wood-Ljungdahl pathway for ethanol synthesis,and showing a strategy for increasing ethanol production in acetogenicClostridia by aldehyde ferredoxin oxidoreductase (AOR) in a host strainthat is attenuated in its ability to produce acetate and has increasedNADPH-dependent alcohol dehydrogenase activity, in accordance with theinvention;

FIG. 8 is a diagram of the butanol and butyrate biosynthesis pathway inC. carboxidivorans and the corresponding genes catalyzing the conversionof acetyl-CoA to butanol and butyrate showing a strategy for increasingbutanol production, in accordance with the invention.

DETAILED DESCRIPTION

The present invention is directed to novel genetic sequences coding foracetogenic Clostridia micro-organisms that produce ethanol and acidsfrom syngas comprising CO, CO2, H2, or mixtures thereof.

Several species of acetogenic Clostridia that produce C₂-C₆ alcohols andacids via the Wood-Ljungdahl pathway have been characterized: C.ragsdalei, C. ljungdahlii, C. carboxydivorans, and C. autoethanogenum.The genomes of three of these microorganisms were sequenced in order tolocate and modify the portions of the genome that code for the enzymesof interest.

The genes that code for enzymes in the Wood-Ljungdahl metabolic pathwayand ethanol synthesis identified in the C. ragsdalei genome arepresented in Table 1. The first column identifies the pathway associatedwith each gene. The gene identification numbers indicated in the secondcolumn correspond to the numbers representing the enzymes involved inthe metabolic reactions in the Wood-Ljungdahl pathway shown in FIG. 1and FIG. 2.

TABLE 1 Clostridium ragsdalei genes used in metabolic engineeringexperiments. Gene EC Pathway ID Gene Name number ORF ID Copy IDDescription Wood- 1 Carbon Monoxide 1.2.2.4 RCCC00183 CODH_1 COoxidation Ljungdahl 2 Dehydrogenase RCCC01175 CODH_2 CO oxidation 3RCCC01176 CODH_3 CO oxidation 4 RCCC02026 CODH_4 CO oxidation 5RCCC03874 CODH_5 CO oxidation 6 Carbon Monoxide 1.2.99.2 RCCC03862cooS/acsA bifunctional Dehydrogenase/Acetyl- CODH/ACS CoA Synthaseenzyme, carbon fixation 7 Formate Dehydrogenase 1.2.1.2 RCCC00874 FDH_1Methyl branch 8 RCCC03324 FDH_2 carbon fixation 9 Formyltetrahydrofolate6.3.4.3 RCCC03872 FTHFS Methyl branch Synthase carbon fixation 10Methenyltetrahydrofolate 3.5.4.9 RCCC03870 MEC Methyl branchcyclohydrolase carbon fixation 11 Methylenetetrahydrofolate 1.5.1.5RCCC03870 MED Methyl branch dehydrogenase carbon fixation 12Methylenetetrahydrofolate 1.5.1.20 RCCC03868 MER Methyl branch reductasecarbon fixation 13 Methyltransferase 2.1.1.13 RCCC03863 acsE Methylbranch carbon fixation 14 Corrinoid/Iron-sulfur 1.2.99.2 RCCC03864 acsCPart of protein CODH/ACS complex, Large subunit 15 Corrinoid/Iron-sulfur1.2.99.2 RCCC03865 acsD Part of protein CODH/ACS complex, Small subunitEthanol and 16 Acetate Kinase 2.7.2.1 RCCC01717 ACK Acetate acetateproduction production 17 Phospho-transacetylase 2.3.1.8 RCCC01718 PTAAcetate production 18 Tungsten-containing 1.2.7.5 RCCC00020 AOR_1Reduction of aldehyde ferredoxin acetate to oxidoreductase acetaldehyde19 1.2.7.5 RCCC00030 AOR_2 Reduction of acetate to acetaldehyde 201.2.7.5 RCCC01183 AOR_3 Reduction of acetate to acetaldehyde 21Acetyl-CoA Reductase 1.1.1.2 RCCC02715 ADH_1 zinc-containing, NADPH-dependent Acetyl-CoA reductase 22 Alcohol Dehydrogenase 1.1.1.1RCCC01356 ADH_2 two pfam domain: FeAHD and ALDH, AdhE 23 1.1.1.1RCCC01357 ADH_3 two pfam domain: FeADH and ALDH, AdhE 24 1.1.1.1RCCC01358 ADH_4 two pfam domain: FeADH and ALDH, AdhE, fragment (76aa)25 1.1.1.1 RCCC03300 ADH_5 one pfam domain: FeADH 26 1.1.1.1 RCCC03712ADH_6 one pfam domain: FeADH 27 1.1.1.1 RCCC04095 ADH_7 one pfam domain:FeADH 28 1.—.—.— RCCC00004 ADH_8 short chain ADH, multiple copy 291.—.—.— RCCC01567 ADH_9 short chain ADH, multiple copy 30 1.—.—.—RCCC02765 ADH_10 short chain ADH, multiple copy 31 1.—.—.— RCCC02240ADH_11 short chain ADH, multiple copy 32 Aldehyde Dehydrogenase 1.2.1.10RCCC03290 ALDH_1 Acetylating 33 1.2.1.10 RCCC04101 ALDH_2 Acetylating 341.2.1.10 RCCC04114 ALDH_3 Acetylating Hydrogenase 35 Hydrogenase1.12.7.2 RCCC00038 HYD_1 Fe only, H2 production 36 1.12.7.2 RCCC00882HYD_2 Fe only, large subunit, H2 production 37 1.12.7.2 RCCC01252 HYD_3Fe only, H2 production 38 1.12.7.2 RCCC01504 HYD_4 Fe only, H2production 39 1.12.7.2 RCCC02997 HYD_5 Ni—Fe large subunit, H2 oxidationElectron 40 Ferredoxin RCCC00086 carrier 41 RCCC00301 42 RCCC00336 43RCCC01168 44 RCCC01415 45 RCCC01825 46 RCCC02435 47 RCCC02890 48RCCC03063 49 RCCC03726 50 RCCC04003 51 RCCC04147 Electron 52 Pyridinenucleotide- RCCC02615 glutamate transfer disulphide synthase smalloxidoreductases chain, but no large chain next to it 53 RCCC02028 nextto cooF and cooS, probably important for reduced pyridine cofactorgeneration 54 RCCC03071 NADH dehydrogenase, not part of an operon 55Membrane-associated RCCC02027 cooF Between gene electron transfer FeSnumber 4 and protein, cooF gene number 53

Sequence analysis of the C. ljungdahlii genome was conducted. Genescoding for enzymes in the Wood-Ljungdahl pathway, ethanol and acetateproduction, and electron transfer have been identified and locatedwithin the genome. The results are presented in Table 2.

TABLE 2 Clostridium ljungdahlii genes used in metabolic engineeringexperiments. Gene EC Pathway ID Gene Name number ORF ID Copy IDDescription Wood- 1 Carbon Monoxide 1.2.2.4 RCCD00983 CODH_1 COoxidation Ljungdahl 2 Dehydrogenase RCCD00984 CODH_2 CO oxidation 3RCCD01489 CODH_3 CO oxidation 4 RCCD04299 CODH_4 CO oxidation 5 CarbonMonoxide 1.2.99.2 RCCD00972 CODH_ACS bifunctional Dehydrogenase/Acetyl-CODH/ACS CoA Synthase enzyme, carbon fixation 6 Formate Dehydrogenase1.2.1.2 RCCD01275 FDH_1 Methyl branch 7 RCCD01472 FDH_2 carbon fixation8 Formyltetrahydrofolate 6.3.4.3 RCCD00982 FTHFS Methyl branch Synthasecarbon fixation 9 Methenyltetrahydrofolate 3.5.4.9 RCCD00980 MEC Methylbranch cyclohydrolase carbon fixation 10 Methylenetetrahydrofolate1.5.1.5 RCCD00980 MED Methyl branch dehydrogenase carbon fixation 11Methylenetetrahydrofolate 1.5.1.20 RCCD00978 MER Methyl branch reductasecarbon fixation 12 Methyltransferase 2.1.1.13 RCCD00973 MET Methylbranch carbon fixation 13 Corrinoid/Iron-sulfur 1.2.99.2 RCCD00974 COPLPart of protein CODH/ACS complex, Large subunit 14 Corrinoid/Iron-sulfur1.2.99.2 RCCD00975 COPS Part of protein CODH/ACS complex, Small subunitEthanol and 15 Acetate Kinase 2.7.2.1 RCCD02720 ACK Acetate acetateproduction production 16 Phospho-transacetylase 2.3.1.8 RCCD02719 PTAAcetate production 17 Tungsten-containing 1.2.7.5 RCCD01679 AOR_1Reduction of aldehyde ferredoxin acetate to oxidoreductase acetaldehyde18 1.2.7.5 RCCD01692 AOR_2 Reduction of acetate to acetaldehyde 19Acetyl-CoA Reductase 1.1.1.2 RCCD00257 ADH_1 zinc-containing NADPH-dependent Acetyl-CoA Reductase 20 Alcohol Dehydrogenase 1.1.1.1RCCD00167 ADH_2 two pfam domain: FeADh and ALDH, AdhE 21 1.1.1.1RCCD00168 ADH_3 two pfam domain: FeADh and ALDH, AdhE 22 1.1.1.1RCCD02628 ADH_5 one pfam domain: FeADh 23 1.1.1.1 RCCD03350 ADH_7 onepfam domain: FeADh 24 1.—.—.— RCCD00470 ADH_8 short chain ADH, multiplecopy 25 1.—.—.— RCCD01665 ADH_9 short chain ADH, multiple copy 261.—.—.— RCCD01767 ADH_10 short chain ADH, multiple copy 27 1.—.—.—RCCD02864 ADH_11 short chain ADH, multiple copy 28 AldehydeDehydrogenase 1.2.1.10 RCCD02636 ALDH_1 Acetylating 29 1.2.1.10RCCD03356 ALDH_2 Acetylating 30 1.2.1.10 RCCD03368 ALDH_3 AcetylatingHydrogenase 31 Hydrogenase 1.12.7.2 RCCD00346 HYD_1 Ni—Fe large subunit,H2 oxidation 32 1.12.7.2 RCCD00938 HYD_2 Ni—Fe small subunit, H2oxidation 33 1.12.7.2 RCCD01283 HYD_3 Fe only, large subunit, H2production 34 1.12.7.2 RCCD01700 HYD_4 Fe only, H2 production 351.12.7.2 RCCD02918 HYD_5 Fe only, H2 production 36 1.12.7.2 RCCD04233HYD_6 Fe only, H2 production Electron 37 Ferredoxin RCCD00424 carrier 38RCCD01226 39 RCCD01932 40 RCCD02185 41 RCCD02239 42 RCCD02268 43RCCD02580 44 RCCD03406 45 RCCD03640 46 RCCD03676 47 RCCD04306 Electron48 Pyridine nucleotide- RCCD00185 glutamate transfer disulphide synthasesmall oxidoreductases chain, but no large chain next to it 49 RCCD01487next to cooF and cooS, probably important for reduced pyridine cofactorgeneration 50 RCCD00433 NADH dehydrogenase, not part of an operon 51Membrane-associated RCCD01488 cooF Between gene electron transfer FeSnumber 3 and protein, cooF gene number 49

Similarly, the genome of C. carboxydivorans was sequenced, and genescoding for the enzymes in the Wood-Ljungdahl pathway and ethanol andacetate synthesis were identified and located. The results are presentedin Table 3.

TABLE 3 Clostridium carboxidivorans genes used in metabolic engineering.Gene EC Pathway ID Gene Name number ORF ID Copy ID Description Wood- 1Carbon Monoxide 1.2.2.4 RCCB04039 CODH_1 CO oxidation Ljungdahl 2Dehydrogenase RCCB01154 CODH_2 CO oxidation 3 RCCB02478 CODH_3 COoxidation 4 RCCB03963 CODH_4 CO oxidation 5 RCCB04038 CODH_5 COoxidation 6 Carbon Monoxide 1.2.99.2 RCCB04293 CODH_ACS bifunctionalDehydrogenase/Acetyl- CODH/ACS CoA Synthase enzyme, carbon fixation 7Formate Dehydrogenase 1.2.1.2 RCCB05406 FDH_1 Methyl branch 8 RCCB01346FDH_2 carbon fixation 9 Formyltetrahydrofolate 6.3.4.3 RCCB04040 FTHFSMethyl branch Synthase carbon fixation 10 Methenyltetrahydrofolate3.5.4.9 RCCB04042 MEC Methyl branch cyclohydrolase carbon fixation 11Methylenetetrahydrofolate 1.5.1.5 RCCB04042 MED Methyl branchdehydrogenase carbon fixation 12 Methylenetetrahydrofolate 1.5.1.20RCCB04044 MER Methyl branch reductase carbon fixation 13Methyltransferase 2.1.1.13 RCCB04294 MET Methyl branch carbon fixation14 Corrinoid/Iron-sulfur 1.2.99.2 RCCB04049 COPL Part of proteinCODH/ACS complex, Large subunit 15 Corrinoid/Iron-sulfur 1.2.99.2RCCB04047 COPS Part of protein CODH/ACS complex, Small subunit Ethanoland 16 Acetate Kinase 2.7.2.1 RCCB05249 ACK Acetate acetate productionproduction 17 Phospho-transacetylase 2.3.1.8 RCCB02481 PTA Acetateproduction 18 Tungsten-containing 1.2.7.5 RCCB00063 AOR_1 Reduction ofaldehyde ferredoxin acetate to oxidoreductase acetaldehyde 19 AlcoholDehydrogenase 1.1.1.2 RCCB03584 ADH_1 zinc-ADH 20 1.1.1.1 RCCB03870ADH_2 two pfam domain: FeADH and ALDH, AdhE 21 1.1.1.1 RCCB05675 ADH_3truncated, AdhE 22 1.1.1.1 RCCB00958 ADH_5 one pfam domain: FeADH 231.1.1.1 RCCB04489 ADH_6 one pfam domain: FeADH 24 1.1.1.1 RCCB04503ADH_7 one pfam domain: FeADH 25 1.—.—.— RCCB02465 ADH_9 short chain ADH,multiple copy 26 1.—.—.— RCCB05551 ADH_10 short chain ADH, multiple copy27 Aldehyde Dehydrogenase 1.2.1.10 RCCB02403 ALDH_1 Acetylating 281.2.1.10 RCCB02561 ALDH_2 Acetylating 29 1.2.1.10 RCCB04031 ALDH_3Acetylating Hydrogenase 30 Hydrogenase 1.12.7.2 RCCB02249 HYD_1 Ni—Felarge subunit, H2 oxidation 31 1.12.7.2 RCCB01319 HYD_2 Fe only, H2production 32 1.12.7.2 RCCB01405 HYD_3 Fe only, H2 production 331.12.7.2 RCCB01516 HYD_4 Fe only, large subunit, H2 production 341.12.7.2 RCCB03483 HYD_5 Fe only, H2 production 35 1.12.7.2 RCCB05411HYD_6 Fe only, large subunit, H2 production Electron 36 FerredoxinRCCB00234 carrier 37 RCCB00345 38 RCCB01260 39 RCCB01334 40 RCCB01775 41RCCB01960 42 RCCB01972 43 RCCB02618 44 RCCB02638 45 RCCB02836 46RCCB02853 47 RCCB03023 48 RCCB03191 49 RCCB03278 50 RCCB03452 51RCCB03596 52 RCCB03762 53 RCCB03972 54 RCCB04165 55 RCCB04383 56RCCB04571 57 RCCB04585 58 RCCB05780 59 RCCB05975 60 RCCB06304 61RCCB06305 Electron 62 Pyridine nucleotide- RCCB00442 NADH transferdisulphide dehydrogenase, oxidoreductases not part of an operon 63RCCB01674 NADH dehydrogenase, not part of an operon 64 RCCB03510 next tocooF and cooS, probably important for reduced pyridine cofactorgeneration 65 RCCB00586 NADH dehydrogenase, not part of an operon 66RCCB04795 NADH: ferredoxin oxidoreductasen not part of an operon 67Membrane-associated RCCB03509 cooF Between gene electron transfer FeSnumber 2 and protein, cooF gene number 64

Genes that code for enzymes in the electron transfer pathway includecarbon monoxide dehydrogenase, Enzyme Commission number (EC 1.2.2.4).Five separate open reading frame (ORF) sequences were identified in C.ragsdalei and C. ljungdahlii, and six were identified in the C.carboxidivorans genome for the carbon monoxide dehydrogenase enzyme.

FIG. 3 is a diagram of carbon-monoxide dehydrogenase operon 300. Thegene order within operon 300 is highly conserved in all three species ofacetogenic Clostridia, and comprises the genes coding for the carbonmonoxide dehydrogenase (cooS) (Gene ID 4, Tables 1, 2, and 3), followedby the membrane-associated electron transfer FeS protein (cooF) (Gene ID55, Table 1; Gene ID 51, Table 2; Gene ID 67, Table 3), in turn,followed by ferredoxin oxidoreductase (FOR).

A comparison was conducted of the genetic sequence found in the operonof FIG. 3 across the three species of acetogenic Clostridia. The cooSgene had 98% identity between C. ragsdalei and C. ljungdahlii, 84%identity between C. carboxydivorans and C. ragsdahlii, and 85% identitybetween C. carboxydivorans and C. ljungdahlii. The cooF gene had 98%identity between C. ragsdalei and C. ljungdahlii, 80% identity betweenC. carboxydivorans and C. ragsdalei, and 81% identity between C.carboxydivorans and C. ljungdahlii. The FOR gene had 97% identitybetween C. ragsdalei and C. ljungdahlii, 77% identity between C.carboxydivorans and C. ragsdalei, and 77% identity between C.carboxydivorans and C. ljungdahlii.

Six hydrogenase (EC 1.12.7.2) ORF sequences were identified in thegenome of each of the acetogenic Clostridium species.

Twelve ferredoxin biosynthesis genes (Gene ID 40-51) were identified inthe C. ragsdalei genome. Eleven ferredoxin biosynthesis genes (Gene ID37-47, Table 2) were found in C. ljungdahlii, and twenty-six (Gene ID36-61, Table 3) were found in C. carboxidivorans.

Three genes coding for ferredoxin oxidoreductase enzymes were found inthe C. ragsdalei genome that contain both a ferredoxin and nicotinamidecofactor binding domain. The ORF Sequence ID numbers (Table 1) for thesegenes are: RCCCO2615; RCCCO2028; and RCCCO3071. The key gene formetabolic engineering, RCCCO2028, is part of the cooS/cooF operon, alsoshown in FIG. 3. Similarly, three genes coding for ferredoxinoxidoreductase (FOR) enzymes were found in the C. ljungdahlii genome.Each of these genes code for both the ferredoxin and cofactor bindingdomains. The ORF Sequence ID numbers for these genes are: RCCD00185;RCCD01847; and RCCD00433 (Table 2). The key gene RCCD01847, is part ofthe cooF/cooS operon shown in FIG. 3.

Five genes were found in the C. carboxidivorans genome that contain boththe ferredoxin and cofactor binding domains. The ORF Sequence ID numbers(Table 3) for these genes are: RCCB00442; RCCB01674; RCCB03510;RCCB00586; and RCCB 04795. The potentially key gene for modulatingelectron flow is RCCB03510, which is part of the cooF/cooS operon (FIG.3).

The genes encoding AR (Gene ID 21, Table 1; Gene ID 19, Table 2) weresequenced in C. ragsdalei and C. ljungdahlii. A high degree of geneconservation is observed for the acetyl CoA reductase gene in C.ragsdalei and C. ljungdahlii. Furthermore, in both micro-organisms, theenzyme exhibits a high degree of homology. The sequence of the acetylCoA gene in C. ragsdalei and C. ljungdahlii was compared and found tohave a 97.82% identity.

Further, the functionality of the gene (including the promoter) encodingfor acetyl CoA reductase was tested. The gene was amplified by PCR,transferred into shuttle vector pCOS52 and ligated into the EcoRI siteto form pCOS54. The vector contained the entire acetyl-CoA reductasegene and its promoter on a high-copy plasmid. pCOS52 contained the samebackbone vector as pCOS54 but lacked the AR gene. pCOS52 was used as thecontrol plasmid in functional assays to determine expression of the ARgene in E. coli to confirm the Clostridial gene function. The resultsconfirmed the function of the acetyl CoA reductase gene.

The functional assay consisted of adding cells harvested at the giventime points to a reaction buffer containing NADPH and acetone as thesubstrate. Spectrophotometric activity (conversion of NADPH to NADP+)was measured at 378 nm and compared to a standard curve to determinetotal activity level. Specific activity was determined using 317 mg/gramof dry cell weight at an OD measurement of 1.

The genes encoding the PTA-ACK operon (Gene IDs 16-17, Tables 1 and 3;Gene IDs 15-16, Table 2) and its promoter were sequenced in C.ragsdalei, C. ljungdahlii, and C. carboxydivorans. The functionality ofthe operon was confirmed, and it was demonstrated that downregulation ofthe operon increases the ethanol to acetate production ratio.Downregulation involves decreasing the expression o the transcription ofthe 2-gene operon via promoter modification through site-directedmutagenesis. Such downregulation leads to a decrease in mRNA, leading toa decrease in protein production and a corresponding decrease in theability of the strain to produce acetate. Such downregulation can beachieved via the method described in Example 2.

Additionally, a comparison was conducted of the genetic sequence foundin the PTA-ACK operon across three species of acetogenic Clostridia. ThePTA gene had 97% identity between C. ragsdalei and C. ljungdahlii, 78%identity between C. carboxydivorans and C. ragsdalei, and 79% identitybetween C. ljungdahlii and C. carboxydivorans. The ACK gene had 96%identity between C. ragsdalei and C. ljungdahlii, 78% between C.carboxydivorans and C. ragsdalei, and 77% between C. carboxydivorans andC. ljungdahlii.

Key genes to promote production of ethanol in C. ragsdalei include: SEQID NO 1 (Gene ID Nos. 4, 55, 53, Table 1) the gene sequence, includingthe experimentally determined promoter region, for carbon monoxidedehydrogenase, coos, electron transfer protein cooF, and the NADHdependent ferredoxin oxidoreductase (FOR);

SEQ ID NO 2 (Gene ID Nos. 17, 16, Table 1), the gene sequence, includingthe experimentally determined promoter region, for ACK and PTA;

SEQ ID NO 3 (Gene ID No. 6, Table 1), the gene sequence, including theexperimentally determined promoter region, for the acetyl CoA reductase;

Sequence Listing

C. ragsdalei gene sequences (Table 1)

>SEQ ID NO. 1: (cooS, cooF, NADH:Ferredoxin Oxidoreductase operon (includesSTOP), Gene ID Nos. 4, 55, 53)TATTATATCAATATAGAATAATTTTCAATCAAATAAGAATTATTTTATATTTTATATTGACAAGGAAACCGAAAAGGTTTATATTATTGTTATTGGATAACAATTATTTTTTAGTTAGTTGTACTTGTAAATAAATAGTATTAATTAATACTATTAAACTATTACAGTTTTTGATTCTTAGTATAAGTATTCTTAGTATCTTTAGCACTTAGAATACGTTATCCTTTAGGAGAATAATCCTAATCAGTAATTTTAATAATTTAATAGTATACTTAAATAGTATAGTTTGGAGGTTTTATTATGTCAAATAACAAAATTTGTAAGTCAGCAGATAAGGTACTTGAAAAGTTTATAGGTTCTCTAGATGGTGTAGAAACTTCTCATCATAGGGTAGAAAGCCAAAGTGTTAAATGTGGTTTTGGTCAGCTAGGAGTCTGCTGTAGACTCTGTGCAAACGGTCCCTGCAGAATAACACCTAAAGCTCCAAGAGGAGTATGTGGTGCTAGTGCTGATACCATGGTTGCAAGAAACTTTCTTAGAGCTGTAGCTGCCGGCAGTGGATGTTATATCCATATAGTCGAAAATACAGCTAGAAACGTAAAATCAGTAGGTGAAACCGGCGGAGAGATAAAAGGAATGAATGCTCTCAACACCCTAGCAGAAAAACTTGGTATAACAGAATCTGACCCACATAAAAAAGCTGTACTAGTAGCTGTGCCGTATTAAAGGACTTATACAAACCAAAATTCGAAAAAATGGAAGTTATAAATAAATTAGCTTATGCACCTAGACTAGAAAATTGGAACAAATTAAATATAATGCCTGGCGGTGCAAAATCAGAAGTTTTTGATGGTGTAGTAAAAACTTCTACAAATCTAAACAGCGACCCTGTAGATATGCTTCTAAATTGTTTAAAACTTGGAATATCCACTGGGATTTACGGACTTACCCTTACAAATTTATTAAATGACATAATTTTAGGTGAACCTGCTATAAGACCTGCAAAAGTTGGTTTTAAAGTTGTAGATACGGATTATATAAATTTGATGATAACAGGCCACCAGCACTCCATGATTGCCCACCTTCAAGAAGAACTTGTAAAACCTGAAGCTGTAAAAAAAGCCCAAGCAGTTGGTGCTAAAGGATTCAAACTAGTTGGATGTACCTGTGTCGGACAGGATTTACAGTTAAGAGGTAAATACTATACTGATGTTTTCTCCGGTCATGCAGGAAATAACTTTACAAGTGAAGCCTTAATAGCAACTGGAGGTATAGATGCAATAGTATCTGAATTTAACTGTACTCTTCCTGGCATCGAGCCAATAGCTGATAAGTTCATGGTTAAAATGATATGCCTAGATGACGTTTCTAAAAAATCAAATGCAGAATATGTAGAATACTCTTTTAAAGATAGAGAAAAAATAAGCAACCATGTTATAGATACGGCTATTGAAAGTTATAAGGAAAGAAGATCTAAAGTTACAATGAATATTCCTAAAAACCATGGCTTTGATGACGTCATAACAGGTGTAAGTGAAGGTTCCTTAAAATCCTTCTTAGGCGGAAGTTGGAAACCTCTTGTAGACTTAATTGCTGCTGGAAAAATTAAAGGTGTTGCTGGAATAGTAGGTTGTTCAAACTTAACTGCCAAAGGTCACGATGTATTTACAGTAGAACTTACAAAAGAACTCATAAAGAGAAATATAATTGTACTTTCTGCAGGTTGTTCAAGTGGTGGACTTGAAAATGTAGGACTTATGTCTCCAGGAGCTGCTGAACTTGCAGGAGATAGCTTAAAAGAAGTATGTAAGAGCCTAGGTATACCACCTGTACTAAATTTTGGTCCATGTCTTGCTATTGGAAGATTGGAAATTGTAGCAAAAGAACTAGCAGAATACCTAAAAATAGATATTCCACAGCTTCCACTTGTGCTTTCTGCACCTCAATGGCTTGAAGAACAAGCATTGGCAGATGGAAGTTTTGGTCTTGCCCTTGGATTACCACTTCACCTTGCTATATCTCCTTTCATTGGTGGAAGCAAAGTGGTAACAAAAGTTTTATGTGAAGATATGGAAAATCTAACAGGCGGCAAGCTTATAATAGAAGACGATGTAATAAAAGCTGCAGATAAATTAGAAGAAACCATACTTGCAAGAAGGAAAAGCTTAGGTCTTAATTAAATGAAAAGAATAATGATAAATAAGGATTTATGTACCGGATGCTTAAATTGTACTTTAGCTTGTATGGCAGAACACAATGAAAATGGGAAATCTTTTTATGATCTGGATCTCAGCAATAAATTTCTTGAAAGTAGAAATCATATATCTAAAGATGATAATGGAAACAAGCTTCCTATATTTTGCCGTCACTGTGACGAACCTGAGTGCGTAATGACATGTATGAGCGGTGCCATGACTAAAGATCCTGAAACTGGTATAGTATCCTATGATGAGCATAAATGTGCCAGCTGCTTTATGTGCGTCATGTCCTGTCCTTATGGAGTATTGAAACCAGATACTCAGACCAAAAGTAAAGTAGTTAAATGTGACCTGTGTGGTGACAGAGATACACCTAGATGCGTTGAAAATTGTCCAACAGAAGCAATTTATATTGAAAAGGAGGCAGATCTCCTATGAATGAGTGGTTTAACAATAAAAATATTTTTTCACACAAAATATGTAATAATAGGAGCCAGTGCTGCTGGAATAAATGCTGCTAAAACTTTAAGAAAGTTAGATAAATCCTCCAAAATAACTATTATTTCAAAGGATGATGCAGTTTATTCAAGATGTATACTCCACAAAGTACTTGAGGGAAGTAGAAATTTAGATACCATAAATTTTGTAGATTCTGATTTCTTTGAAAAAAATAATATAGAATGGATAAAAGATGCAGATGTAAGCAATATTGATATTGACAAGAAAAAAGTCTTACTTCAAGACAACAGCAGCTTCAAATTTGACAAGCTCCTTATAGCTTCTGGTGCTTCCTCCTTTATTCCCCCAGTTAAAAAATTAAGAGAAGCTAAAGGAGTGTACTCCCTTAGAAATTTTGAAGATGTAACTGCTATACAAGACAAACTTAAAAACGCAAAACAAGTGGTAATACTTGGTGCAGGTCTTGTAGGAATTGATGCACTTTTAGGTCTTATGGTGAAAAATATAAAGATTTCAGTTGTAGAAATGGGAGATAGGATTCTCCCCCTTCAACTGGACAAAACTGCATCCACTATATATGAAAAGTTGTTAAAAGAAAAAGGTATAGATGTCTTTACTTCAGTTAAATTGGAAGAGGTAGTTTTAAATAAAGACGGAACTGTAAGTAAAGCAGTACTATCAAATTCAACTTCTATAGATTGCGATATGATAATAGTTGCTGCTGGTGTTAGACCAAATGTAAGCTTTATAAAAGACAGCAGGATAAAAGTTGAAAAAGGCATTGTCATAGACAAACATTGTAAAACCACTGTAGATAATATATATGCTGCAGGAGATGTTACTTTTACTGCTCCTATATGGCCTATAGCTGTAAAGCAGGGAATAACTGCTGCTTACAACATGGTAGGTATAAATAGAGAATTACATGACACTTTTGGCATGAAGAACTCAATGAATTTATTTAACCTTCCATGCGTATCCCTTGGTAATGTAAATATAGCAGATGAAAGTTATGCTGTTGATACATTAGAAGGAGATGGAGTTTATCAAAAAATAGTTCACAAAGATGGAGTAATCTACGGTGCACTTCTAGTTGGAGATATATCTTACTGCGGCGTACTAGGATATCTCATAAAAAATAAAGTAAATATAAGCAATATCCATAAAAATATTTTTGACATAGATTATTCTGATTTTTACAATGTTGAAGAAGATGGACAATATAGTTATCAATTGAGGTAASEQ ID NO. 2: (PTA-ACK operon (includes STOP), Gene ID Nos. 17, 16)GCATACTGATTGATTATTTATTTGAAAATGCCTAAGTAAAATATATACATATTATAACAATAAAATAAGTATTAGTGTAGGATTTTTAAATAGAGTATCTATTTTCAGATTAAATTTTTACTTATTTGATTTACATTGTATAATATTGAGTAAAGTATTGACTAGTAAAATTTTGTGATACTTTAATCTGTGAAATTTCTTAGCAAAAGTTATATTTTTGAATAATTTTTATTGAAAAATACAACTAAAAAGGATTATAGTATAAGTGTGTGTAATTTTGTGTTAAATTTAAAGGGAGGAAATAAACATGAAATTGATGGAAAAAATTTGGAATAAGGCAAAGGAAGACAAAAAAAAGATTGTCTTAGCTGAAGGAGAAGAAGAAAGAACTCTTCAAGCTTGTGAAAAAATAATTAAAGAAGGTATTGCAAATTTAATCCTTGTAGGGAATGAAAAGGTAATAGAGGAGAAGGCATCAAAATTAGGCGTAAGTTTAAATGGAGCAGAAATAGTAGATCCAGAAACCTCGGATAAACTAAAAAAATATGCAGATGCTTTTTATGAATTGAGAAAGAAGAAGGGAATAACACCAGAAAAAGCGGATAAAATAGTAAGAGATCCAATATATTTTGCTACGATGATGGTTAAGCTTGGAGATGCAGATGGATTGGTTTCAGGTGCAGTGCATACTACAGGTGATCTTTTGAGACCAGGACTTCAAATAGTAAAGACAGCTCCAGGTACATCAGTAGTTTCCAGCACATTTATAATGGAAGTACCAAATTGTGAATATGGTGACAATGGTGTACTTCTATTTGCTGATTGTGCTGTAAATCCATGCCCAGATAGTGATCAATTGGCTTCAATTGCAATAAGTACAGCAGAAACTGCAAAGAACTTATGTGGAATGGATCCAAAAGTAGCAATGCTTTCATTTTCTACTAAGGGAAGTGCAAAACACGAATTAGTAGATAAAGTTAGAAATGCTGTAGAAATTGCCAAAAAAGCTAAACCAGATTTAAGTTTGGACGGAGAATTACAATTAGATGCCTCTATCGTAGAAAAGGTTGCAAGTTTAAAGGCTCCTGAAAGTGAAGTAGCAGGAAAAGCAAATGTACTTGTATTTCCAGATCTCCAAGCAGGAAATATAGGTTATAAACTTGTTCAAAGATTTGCAAAAGCTGATGCTATAGGACCTGTATGCCAGGGATTTGCAAAACCTATAAATGATTTGTCAAGAGGATGTAACTCCGATGATATAGTAAATGTAGTAGCTGTAACAGCAGTTCAGGCACAAGCTCAAAAGTAAATGAAAATATTAGTAGTAAACTGTGGAAGTTCATCTTTAAAATATCAACTTATTGATATGAAAGATGAAAGCGTTGTGGCAAAAGGACTTGTAGAAAGAATAGGAGCAGAAGGTTCAGTTTTAACACATAAAGTTAACGGAGAAAAGTTTGTTACAGAGCAGCCAATGGAAGATCATAAAGTTGCTATACAATTAGTATTAAATGCTCTTGTAGATAAAAAACATGGTGTAATAAAAGATATGTCAGAAATATCTGCTGTAGGGCATAGAGTTTTGCATGGTGGAAAAAAATATGCGGCATCCATTCTTATTGATGACAATGTAATGAAAGCAATAGAAGAATGTATTCCATTAGGACCATTACATAATCCAGCTAATATAATGGGAATAGATGCTTGTAAAAAACTAATGCCAAATACTCCAATGGTAGCAGTATTTGATACAGCATTTCATCAGACAATGCCAGATTATGCTTATACTTATGCAATACCTTATGATATATCTGAAAAGTATGATATCAGAAAATATGGTTTTCATGGAACTTCTCATAGATTCGTTTCAATTGAAGCAGCCAAGTTGTTAAAGAAAGATCCAAAAGATCTTAAGCTAATAACTTGTCATTTAGGAAATGGAGCTAGTATATGTGCAGTAAACCAGGGAAAAGCAGTAGATACAACTATGGGACTTACTCCCCTTGCAGGACTTGTAATGGGAACTAGATGTGGTGATATAGATCCAGCTATAATACCATTTGTAATGAAAAGAACAGGTATGTCTGTAGATGAAATGGATACTTTAATGAACAAAAAGTCAGGAATACTTGGAGTATCAGGAGTAAGCAGCGATTTTAGAGATGTAGAAGAAGCTGCAAATTCAGGAAATGATAGAGCAAAACTTGCATTAAATATGTATTATCACAAAGTTAAATCTTTCATAGGAGCTTATGTTGCAGTTTTAAATGGAGCAGATGCTATAATATTTACAGCAGGACTTGGAGAAAATTCAGCTACTAGCAGATCTGCTATATGTAAGGGATTAAGCTATTTTGGAATTAAAATAGATGAAGAAAAGAATAAGAAAAGGGGAGAAGCACTAGAAATAAGCACACCTGATTCAAAGATAAAAGTATTAGTAATTCCTACAAATGAAGAACTTATGATAGCTAGGGATACAAAAGAAATAGTTGAAAAT AAATAASEQ ID NO. 3: (ORF RCCC02715, P11,NADPH-SADH (includes STOP), Gene ID No. 6)ATGAAAGGTTTTGCAATGTTAGGTATTAACAAGTTAGGATGGATTGAAAAGAAAAACCCAGTACCAGGTCCTTATGATGCGATTGTACATCCTCTAGCTGTATCCCCATGTACATCAGATATACATACGGTTTTTGAAGGAGCACTTGGTAATAGGGAAAATATGATTTTAGGTCACGAAGCTGTAGGTGAAATAGCTGAAGTTGGCAGTGAAGTTAAAGATTTTAAAGTTGGCGATAGAGTTATCGTACCATGCACAACACCTGACTGGAGATCCTTAGAAGTCCAAGCTGGTTTTCAACAGCATTCAAACGGTATGCTTGCAGGATGGAAGTTTTCCAATTTTAAAGACGGTGTATTTGCAGATTACTTTCATGTAAACGATGCAGATATGAATCTTGCAATACTTCCAGATGAAATACCTTTAGAAAGTGCAGTTATGATGACAGACATGATGACTACTGGTTTTCATGGGGCAGAACTTGCTGACATAAAAATGGGTTCCAGTGTTGTCGTAATTGGTATAGGAGCTGTTGGATTAATGGGAATAGCCGGTTCCAAACTTCGAGGAGCAGGTAGAATTATCGGTGTTGGAAGCAGACCCGTTTGTGTTGAAACAGCTAAATTTTATGGAGCAACTGATATTGTAAATTATAAAAATGGTGATATAGTTGAACAAATAATGGACTTAACTCATGGTAAAGGTGTAGACCGTGTAATCATGGCAGGCGGTGGTGCTGAAACACTAGCACAAGCAGTAACTATGGTTAAACCTGGCGGCGTAATTTCTAACATCAACTACCATGGAAGCGGTGATACTTTGCCAATACCTCGTGTTCAATGGGGCTGCGGCATGGCTCACAAAACTATAAGAGGAGGGTTATGTCCCGGCGGACGTCTTAGAATGGAAATGCTAAGAGACCTTGTTCTATATAAACGTGTTGATTTGAGCAAACTTGTTACTCATGTATTTGATGGTGCAGAAAATATTGAAAAGGCCCTTTTGCTTATGAAAAATAAGCCAAAAGATTTAATTAAATCAGTAGTTACA TTCTAA

Using detailed genomic information, the acetogenic Clostridiamicro-organisms have been metabolically engineered to increase thecarbon and electron flux through the biosynthetic pathways for ethanoland butanol, while simultaneously reducing or eliminating carbon andelectron flux through the corresponding acetate and butyrate formationpathways, in accordance with the present invention. For this purpose,the activities of key genes encoding for enzymes in the pathway havebeen modulated. In one embodiment, gene expression of key alcoholproducing enzymes is increased by increasing the copy number of thegene. For example, a key carbon monoxide dehydrogenase operon (FIG. 3)and the associated electron transfer proteins, including acetyl CoAreductase and aldehyde ferredoxin oxidoreductase are duplicated withinthe genome of the modified organism. In one embodiment, theseduplications are introduced into strains having knocked out orattenuated acetate production to further channel electrons into theethanol or butanol production pathway. In another embodiment a knockoutstrategy is applied to strains of acetogenic Clostridia that, when grownon syngas, produce more complex mixtures of alcohols and acids, such asethanol, butanol and hexanol and their corresponding carboxylic acids.

In one embodiment, vectors to be used for the transfer of acetogenicClostridia cloned genes from cloning vehicles to parent acetogenicClostridia strains are constructed using standard methods (Sambrook etal., 1989). All gene targets used in molecular genetics experiments areamplified using high-fidelity polymerase chain reaction (PCR) techniquesusing sequence-specific primers. The amplified genes are next subclonedinto intermediate cloning vehicles, and later recombined inmulti-component ligation reactions to yield the desired recombinantvector to be used in the gene transfer experiments. The vectors containthe appropriate functional features required to carry out the genetransfer experiments successfully and vary depending on the method used.

To transfer the recombinant vectors into recipient acetogenicClostridia, a variety of methods are used. These includeelectroporation, bi-parental or tri-parental conjugation,liposome-mediated transformation and polyethylene glycol-mediatedtransformation. Recombinant acetogenic Clostridia are isolated andconfirmed through molecular biology techniques based on the acquisitionof specific traits gained upon DNA integration.

Example 1

Acetogenic Clostridia contain operon 300, shown in FIG. 3, that consistsof carbon monoxide dehydrogenase 104 (cooS, Gene ID 4, Table 1, Table 2,Table 3), a membrane-associated electron transfer protein (cooF), and aferredoxin oxidoreductase (FOR). Overexpression of carbon monoxidedehydrogenase 104 within the acetogenic Clostridia is known to increaseelectron flow from syngas components to the oxidizeded nucleotidecofactors NAD⁺ and NADP⁺ The increased levels of reduced nucleotidecofactors then stimulate generation of intermediate compounds inWood-Ljungdahl pathway 100.

In one embodiment, operon 300 is amplified using long-PCR techniqueswith primers that are designed to anneal to a region 200 nucleotides(nt) upstream of the carbon monoxide dehydrogenase gene and 200 ntdownstream of the ferredoxin oxidoreductase gene. The total region isabout 3.8 kilobase pairs. The amplified DNA is cloned directly intosuitable plasmid vectors specifically designed to ligate PCR productssuch as pGEM T easy (Promega, Madison, Wis.) or pTOPO (Invitrogen,Carlsbad, Calif.). The ends of the PCR product contain engineeredrestriction sites to facilitate later cloning steps. The operon 300 issubcloned into a vector that already contains cloned chromosomal C.ragsdalei or other acetogenic Clostridial DNA to allow chromosomalintegration at a neutral site.

Example 2

Because carboxylic acids compete with alcohols for electrons, decreasingacid production allows more electrons to flow down thealcohol-production pathway from the CoA intermediate directly to thealcohol. Acetogenic Clostridia contain genes for phospho-transacetylaseenzyme (Gene ID 17, Tables 1 and 3; Gene ID 16, Table 2) that convertsacetyl-CoA to acetyl-phosphate and acetate kinase (Gene ID 16, Table 1)that converts acetyl-phosphate 218 to acetate 214. In one embodiment,genetic modifications to delete all or part of the genes for bothenzymes and knock out or attenuate production of acetate are made asshown in FIG. 5.

Using PCR and other standard methods, a recombinant vector containingtwo large non-contiguous segments of DNA is generated. Upon replacementof the native gene by the recombinant vector gene, the Clostridialstrain contains no phosphotransacetylase or acetate kinase activities asshown in FIG. 5 by X 504 and X 502, respectively.

Modulation of the common promoter region, P* 506 to attenuate geneexpression of phosphotransacetylase 508 and acetate kinase 510 andsubsequent acetate production are carried out by generating a series ofrecombinant vectors with altered promoter regions. The vector series isconstructed by site-directed mutagenesis.

Additionally, down-regulation of the 2-gene operon containing pta/ackgenes is performed by site-directed mutagenesis of the promoter region.A decrease in RNA polymerase binding leads to a decrease intranscriptional activity off of the pta/ack promoter and in turn lead toa decrease in protein activity. The end result is a decrease in acetateproduction since the intermediates are produced at a lower rate and morecarbon from acetyl-CoA goes towards ethanol production. A promoter probeassay using a reporter group that is easily quantitated has beendeveloped to measure relative promoter strength of the pta/ack promoterin vivo. After site-directed mutagenesis is performed, which impartssingle and multiple lesions over a 200 base pair region, strains thathave decreased promoter activity are isolated such that a series ofstrains with 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10% and 0% activityof the native promoter in the assay are isolated and tested inrecombinant Clostridia strains.

Example 3

In vivo, the acetyl CoA enzyme designated in 102 and FIG. 5 converts theCoenzyme A (CoA) form of a carbon moiety, such as acetyl-CoA 102 orbutyrl-CoA directly to its corresponding alcohol. Thermodynamically,direct conversion from the CoA form to the alcohol requires transfer offour electrons, and is a more efficient way to generate the alcohol,compared to the two-step conversion of the carboxylic acid to thecorresponding alcohol. For example, as shown in FIG. 6, the two stepconversion requires that acetate 214, first be converted to its aldehydeform (acetaldehyde, 604), and then to the corresponding alcohol, ethanol216. Thus, increasing AR activity, portrayed by the vertical arrow 602is desirable for increasing alcohol production, and increasing theselectivity of the process by increasing the ratio of alcohol to acid.

In one embodiment, AR activity in acetogenic Clostridia is increased byamplifying the gene in vitro using high-fidelity PCR and inserting theduplicated copy of the gene into a neutral site in the chromosome usingstandard molecular genetic techniques. After gene replacement of thevector, the chromosome contains two copies of the AR. Confirmation ofgenereplacement followed by gene expression studies of the recombinantstrain are performed and compared to the parent strain.

In other embodiments a similar strategy is used to increase theenzymatic activity of adhE-type alcohol dehydrogenases, short-chainalcohol-dehydrogenases and primary Fe-containing alcohol dehydrogenases.

Example 4

Under some conditions, Clostridia need to obtain additional energy inthe form of adenosine triphosphate production (ATP) causing the cells totemporarily increase the production of acetate 214 from acetyl-CoA 102.The net reaction is 1 ATP from ADP+P, through acetyl-phosphate. Acetateproduction is advantageous to the syngas fermentation process at low tomoderate acetic acid concentrations, because it allows the cells toproduce more energy and remain robust. However, too much free aceticacid causes dissipation of the transmembrane ion gradient used as theprimary ATP generation source and therefore becomes detrimental to thecells. For industrial production purposes, it is advantageous to convertthe acetate to ethanol to increase ethanol production and reduce theprobability of accumulating too much free acetic acid.

In one embodiment, ethanol production in the double mutant C. ragsdaleistrain is increased by between 10 and 40% as a result of the increasedaldehyde ferredoxin oxidoreductase and AR activities. In anotherembodiment, the ratio of ethanol to acetate produced is increasedbetween 5 and 10 fold, but allows sufficient acetate formation tosupport ATP production needed to meet the energy needs of themicroorgansim.

While the invention has been described with reference to particularembodiments, it will be understood by one skilled in the art thatvariations and modifications may be made in form and detail withoutdeparting from the spirit and scope of the invention.

What is claimed is:
 1. An isolated polynucleotide comprising anucleotide sequence encoding an operon that codes for carbon monoxidedehydrogenase, a membrane-associated electron transfer protein, aferredoxin oxidoreductase, and a promoter, said sequence being at least97% identical to SEQ ID NO.
 1. 2. A vector comprising the polynucleotideof claim
 1. 3. An isolated transformant containing the polynucleotide ofclaim
 1. 4. An isolated transformant carrying the vector of claim
 2. 5.A method of producing ethanol comprising: isolating and purifyinganaerobic, ethanologenic microorganisms carrying the polynucleotide ofclaim 1; fermenting syngas with said microorganisms in a fermentationbioreactor.
 6. A method of increasing ethanologenesis in a microorganismcontaining the nucleotide sequence of claim 1, said method comprising:modifying or duplicating a promoter region of said nucleotide sequenceto increase the activity of the operon of claim 1 or to causeoverexpression of the operon.